This application relates generally to bioreactors and more particularly bioreactors for growing cells in three possible states: static, dynamic, or alternating between static and dynamic states in the same cell culture container(s).
Many kinds of cells, especially hematopoietic stem cells and immunocytes, are very sensitive to shear-stress in the culture. For example, shear-stress can cause the non-specific differentiation and the increased apoptosis in the stem cell culture, which significantly reduces the efficiency of the stem cell expansion and directed differentiation. The higher shear-stress also causes the more release of non-specific proteins in protein expression, in which the protein of interest takes less proportion in the culture and so result in the increased difficulties of protein purification. The static culture has the least shear stress but the cells in static culture normally sit at the bottom of the culture containers, some cells cannot get enough nutrition when cells are at higher density and so not suitable for large scale cell expansion. Some bioreactors, such as NASA's rotation wall vessel (RWV) bioreactor, were designed for reducing the shear-stress. However, these bioreactors have to keep cells in suspension by continuously moving, stirring or/and agitating cells. Once the bioreactor stop running, cells will accumulate or aggregate somewhere of the bottom of or other locations within the cell culture container but are not evenly distributed, which is harmful for most cell growth or at least is not conductive to efficient cell growth. Therefore, though the shear-stress has been reduced in these bioreactors, such a reduced shear-stress has to continuously exert on the cultured cells when these bioreactors are running. No doubt, an intermittent cell agitation, or the suitable alternation between static and dynamic culture, will not only minimize the sheer-stress but also provide cells an ideal metabolic environment. However, it is often very difficult to get cells evenly distributed in the static cell culture following a dynamic culture because of the inertia the moving cells have, while an uneven distribution (such as local accumulation or aggregation) of cells in static culture could be very harmful for cell growth as mentioned above. Our current invention provides some methods to allow cells to be evenly distributed in the static culture following a dynamic culture, so that all three cell culture states can be employed in present disclosed bioreactor system.
Some other bioreactors, such as those invented by Felder and colleagues, the static and dynamic states of the cell cultures are respectively performed in two or more different cell culture containers and the changes between the two states of the cell culture are carried out by transferring cell from one type of cell culture container to the other type of cell culture container. When cells are transferred between containers, the cell losses and damages are inevitable. In our current invention, both static and dynamic cell cultures are carried out in the same cell culture container(s), no cell-transferring between cell culture container is needed during the alternating between the two culture states, and when the cell culture is changed from the dynamic to the static status, cells can be evenly distributed at the bottom of the culture chamber (container) or on the surface of the stirring or supporting materials. Thus, our invention provides cells the best growth condition in both suspension states and static states, and these two states can repeatedly alternate.
Some bioreactors use magnet element (specifically blades or vans) controlled by magnet impeller to agitate culture media to keep cells in suspension status. This kind of bioreactor purposely enhances the shear-stress for the culture requirements of a certain cells and the cells are distributed following the direction of media flow agitated by the impellers when the agitation stops. The higher shear-stress and uneven distribution of the cells in static state following agitation make it significant different from our current invented bioreactor system. In addition to the differences in the application, the bioreactor in our invention does not use blades or canes to be the magnet element, and the magnet beads in our invention actually has no magnetism if they are not in magnet field and they can only gain magnetism when they are placed in magnet field. The magnet beads in our invention are not controlled by impeller but by the changes of magnetic strength affecting the beads' moving.
In one embodiment of our bioreactor, in order to minimize the sheer-stress during the agitation and allow the cells evenly distributed in the bioreactor, the agitators' movement between two ends of the interior cell culture container in combination with the corresponsive inversion of the cell culture container are applied in our current invented system. In comparison, other bioreactors either use agitators or use moving container to keep the cells suspended, and either of them does not allow the cells evenly distributed in the bioreactor following agitation and always exerts higher sheer-stress on cells in the culture. It also needs to be emphasized that the inversion of the cell culture container in our system is not for the cell suspension. Instead, the effects of the movement of the cell culture container on the cells suspension in this embodiment are intended to be minimized in our design, because the movement of the container often tends to result in uneven distribution of the cells in static culture. The inversion of cell culture container in our system mainly provides a suitable condition for the agitators to move from one end to the other end of the interior cell culture container.
In another embodiment of our bioreactor, the changes of cell culture states between static and dynamic are carried out by the changes between vertical rotating culture and horizontal static culture. The speed of the cell culture container rotation and the deceleration of the of the cell culture container from rotating to static state are two critical factors affecting cell distribution in static state. The higher or lower speed and deceleration of the cell culture container rotation can result in uneven distribution of the cells following rotation. In our research, the suitable speed and deceleration were founded.
In our research, we also found that both the time length of the static culture and dynamic culture and the frequency of the alternation between static culture and dynamic culture significantly affect the cell growth and need to be determined based upon the cell type, media, cell density (cell concentration) and cell doubling time (cell growth speed) for the specific cell culture.
In our previous patent application, some of the above embodiments have been mentioned but not clearly shown in the claims. In this application, they are all clearly claimed.